Progress in the Life Sciences depends upon the development of new tools and instruments. Our ability to understand the function of living systems on a cellular and molecular level is greatly enhanced by imaging techniques capable of providing structural and chemical information in vivo. Raman spectroscopy is truly non-invasive, and it could provide significant information on the chemical composition and physical structure of biological tissues. This small-scale (R03) research proposal aims to develop an innovative approach for non-invasive microspectroscopy in order to unambiguously explore changes in chemical composition in cells and tissues. To achieve this goal, the PI will utilize the concept of time-gated Raman imaging, applied for the first time to microscopy to achieve confocal imaging with superior discrimination against the fluorescence background. It is expected that it will provide at least an order of magnitude improvement in the signal-to-noise ratio, which transforms into at least 100 times faster acquisition rates, while improving spatial resolution and eradicating possible ambiguities in resolving complex Raman bands. The first specific aim is building the prototype of a novel instrument and characterizing its performance. The second specific aim is pilot testing of a new instrument for molecular, cellular and tissue imaging for several model systems: 1) collagen self-organization and structural transformation, which leads to bone structure development, 2) photodegradation of retinal pigment epithelium cells, which is considered to be the major cause of blindness, and 3) early caries detection in dental tissues. Many medical research and diagnostic applications require microscopic molecular imaging with minimum invasiveness. Raman microspectroscopy, being a non-invasive, chemically specific technique for molecular imaging, suffers a dramatic drawback due to a strong fluorescent background, which significantly reduces the signal-to-noise ratio and makes it difficult to reveal the fine structure of vibrational bands. The proposed research provides a solution to this problem for microscopic imaging by time-gating the useful signal in a specially designed optical arrangement.